This invention was made under the joint sponsorship of the Chinese University of Hong Kong of Shatin, Hong Kong SAR, and the Hospital Authority, also known as the Prince of Wales Hospital of Shatin, Hong Kong SAR.
This invention relates to the cultivation of keratinocytes and dermal fibroblasts on a biosynthetic membrane and subsequent engraftment of this type of membrane on the neodermis of artificial skin with particular application to humans. This invention also relates to fabricated graft materials.
Prompt wound coverage for protection and stabilization is essential for the treatment of burns. The 3T3 cell-feeder layer technique developed by Rheinwald and Green in the 1970s is the standard method for cultivation of autologous epidermal autograft or so-called cultivated epidermal autograft material (xe2x80x9cCEAxe2x80x9d). A small piece of native skin from the patient can be cultured and expanded to 500 times or more in size within 3-4 weeks. However, the lack of dermis and the fragility of the cultured graft often result in unpredictable grafting success rates ranging from 0%-80%, as reported by Tompkins et al. (1992) and Herzog et al. (1991).
ALLODERM(trademark) and INTEGRA(trademark) human skin substitutes are two currently popular examples of human skin substitute commercially available in the market. INTEGRA(trademark) artificial skin, a brand of artificial skin is sold by Integra LifeScience Corporation of New Jersey, USA, and has been approved by FDA for use in the USA since 1996. Artificial skin is a bilayer biosynthetic sheet comprising porous collagen-glycoaminoglycan integrated with a thin silicone membrane as an outer layer. The use of artificial skin such as INTEGRA(trademark) artificial skin as a biocompatible a cellular dermal replacement in deep and full-thickness burn wounds is well known.
It has been observed that within about 14 to 21 days following the grafting of INTEGRA(trademark) artificial skin, there is full vascularization of the neodermis formed in the INTEGRA(trademark) artificial skin. Thereafter an ultra thin split thickness skin graft must be harvested from a donor site in order to cover the neodermis immediately after the silicone membrane is removed. Substantial research effort has been undertaken in the past to determine the possibility of reliably grafting CEA on the neodermis, since an effective combination of CEA and INTEGRA(trademark) artificial skin should eliminate the second operative stage, the associated pain and scaring, as well as a need for a second donor site, which may not be available in extensively burned patients. If the grafted CEA does not xe2x80x98takexe2x80x99 on the neodermis of INTEGRA(trademark) artificial skin after the silicone membrane is peeled off, it can be replaced by another CEA. Whereas, in the conventional application of INTEGRA(trademark) artificial skin, another split thickness autograft must be harvested from a second or even a third donor site. There have been very limited initial anecdotal reports on experience with such a combination technique, such as Sheridan et al. 1999 and Pandya et al. 1998. At the 10th Congress of International Society For Burn Injuries, November 1998 in Israel, the difficulties with the conventionally cultured graft anchoring onto the neodermis of INTEGRA(trademark) artificial skin were addressed. The exact reasons for such difficulties remain unknown.
LASERSKIN(trademark) artificial skin material is a thin and pliable biosynthetic membrane comprising a 100% benzyl esterified hyaluronic acid derivative suitable for use as a substratum in the growth of skin cells. The recommendation of the manufacturer is to seed human keratinocytes on LASERSKIN(trademark) artificial skin preseeded with irradiated 3T3 cells as feeder layer. When following the manufacturer""s recommendation, it was found that, after the initiation of the formation of keratinocyte colonies, the xenogenic 3T3 cells growing on the LASERSKIN(trademark) artificial skin were less likely to be washed away than those growing on a culture dish as in the conventional Green""s method during each flushing procedure with phosphate-buffered saline. It is believed that the remaining 3T3 cells or debris might have sensitized the host to xenogenic antigen resulting in undesired late graft rejection. What is needed is a cultivation and engraftment procedure with a biocompatible, durable human skin substitute.
The following references, not all of which are prior art for the purposes of a patent application, are hereby made of record and incorporated herein by reference for the purposes described in this text:
1) Rheinwald J, Green H. xe2x80x9cSerial cultivation of strain of human epidermal keratinocytes: The formation of keratinizing colonies from single cells.xe2x80x9d Cell 1975; 6: 331-344.
2) Tompkins R G, Burke J F. xe2x80x9cBurn wound closure using permanent skin replacement material.xe2x80x9d World J Surgery 1992; 16: 47-52.
3) Herzog S R, Meyer A, Woodley D, Peterson H D. xe2x80x9cWound coverage with cultured autologous keratinocytes: Use after burn wound excision, including biopsy follow-up.xe2x80x9d J Trauma 1991; 28: 195-1999.
4) Yannas I V, Burke J F, Orgill D P, Skrabut E M. xe2x80x9cWound tissues can utilize a polymeric template to synthesize a functional extension of skin.xe2x80x9d Science 1982; 215: 174-176.
5) Heimbach D, Letterman A, Burke J et al. xe2x80x9cArtificial dermis for major burns. A multi-center randomized clinical trial.xe2x80x9d Ann Surgery 1988; 208: 313-320.
6) Sheridan R L, Heggerty M, Tompkins R G, Burke J F. xe2x80x9cArtificial skin in massive burns-results at ten years.xe2x80x9d Eur J Plast Surgery 1994; 17: 91-93.
7) Sheridan R L, Tompkins R G. xe2x80x9cSkin substitutes in burns.xe2x80x9d Burns 1999; 25: 97-103.
8) Pandya A N, Woodward B, Parkhouse N. xe2x80x9cThe use of cultured autologous keratinocytes with Integra in the resurfacing of acute burns.xe2x80x9d Plast Reconst Surgery 1998; 102: 825-828.
9) Hultman C S, Brinson G M, Silitharm S, et al. xe2x80x9cAllogenic fibroblasts used to grow cultured epidermal autografts persist in vivo and sensitize the graft recipient for accelerated second-set rejection.xe2x80x9d J Trauma 1996; 41: 51-60.
10) Lam P K, King W K, et al. xe2x80x9cDevelopment and evaluation of a new composite Laserskin graftxe2x80x9d Abstracts of the 10th Congress of International Society For Burn Injuries, Nov. 1-6 1998, Jerusalem, Israel, p. 46.
11) Sato T, Kirimura Y, Mori Y. xe2x80x9cThe co-culture of dermal fibroblasts with human epidermal keratinocytes induces increased prostaglandin E2 production and cyclooxygenase 2 activity in fibroblasts.xe2x80x9d J Investigative Dermatology 1997; 109: 334-339.
12) Pano R J, Rubin J R, Aaronson S A, Mason R. xe2x80x9cKeratinocytes growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.xe2x80x9dJ Clin Invest 1993; 92: 969-977.
13) Burd DAR, Greco R M, Regauer M T, et al. xe2x80x9cHyaluron and wound healing: a new perspective.xe2x80x9d Br J Plast Surgery 1991; 44: 579-584.
14) Feinberg R N, Beebe D C. xe2x80x9cHyaluronate in vasoculogenesis.xe2x80x9d Science 1983; 220: 1177-1179.
15) Alman D G. Practical Statistics for Medical Research Chapman and Hall, London 1991 pp. 211, 260-261.
16) Harris P A, Francesco F di, Barisoni D, Leigh I M, Navsaria H A. xe2x80x9cUse of hyaluronic acid and cultured autologous keratinocytes and fibroblasts in extensive burns.xe2x80x9d Lancet 1999; 353: 35-36.
17) Myers S R, Grady J, Soranzo C, Sander R, et al. xe2x80x9cA hyaluronic acid membrane delivery system for cultured keratinocytes: Clinical take rates in the porcine Kerato-Dermal model.xe2x80x9d J Burn Care Rehabil 1977; 18: 214-222.
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According to the invention, autologous cultured keratinocytes grown on a biocompatible substratum are engrafted on the neodermis of artificial skin covering a wound. Autologous keratinocytes may be cultivated on a commercially available membrane such as LASERSKIN(trademark) artificial skin (available from Fidia Advanced Biopolymers Ltd., Abano Terme (PD), Italy) following pre-seeding with autologous or allogenic dermal fibroblasts. The resultant composite material may then be applied on the neodermis of artificial skin which had been previously engrafted on the patient. The composite material, and specifically Composite Biocompatible Skin Graft (CBSG) material comprises autologous keratinocytes and allogenic or autologous dermal fibroblasts grown on the substratum. A method for fabricating the composite material includes the application of dermal fibroblasts onto the substratum as a feeder layer and then inoculating autologous keratinocytes on the resultant structure. A method for engraftment comprises first applying an artificial skin with a protective silicone membrane on a wound area, thereby allowing vascularization; following vascularization, removing the silicone membrane and engrafting the cultured composite material onto the vascularized artificial skin.
Human fibroblasts used in the cultivation technique according to the invention were found to achieve a role similar to 3T3 cells in the initiation of keratinocyte colonies on LASERSKIN(trademark) artificial skin. Specifically, it was found that the seeding efficacy of human keratinocytes was increased to up to 95%. CBSG containing autologous keratinocytes and autologous dermal fibroblast or allogenic dermal fibroblasts or a combination of autologous and allogenic dermal fibroblasts according to the invention, has been successfully applied to burn patients whose wounds were previously grafted with allografts.
The purpose of this invention is to simplify burn treatment further and eventually to save lives of patients having extensive burns where little or no autologous skin grafts can be repeatedly harvested in a short period of time. With the inventive technique, all dead skin tissue of a patient with extensive bums can be excised within about three to seven days after injury. The wound can be covered with artificial skin, such as Integra(trademark) or any dermal equivalent thereof, and only a small quantity of harvested normal skin would be required to initiate skin culture, which may thereafter be engrafted, according to the invention, on the neodermis of the artificial skin, with resultant lower rejection and infection incidences.
CBSG material according to the invention offers notable advantages. First, the basal proteins (including the early basement membrane proteins such as collagen IV and fibronectin) of the cultured graft are protected from dispase treatment because the keratinocytes are directly cultivated on a pliable LASERSKIN(trademark) artificial skin. This is believed to enhance anchorage of the cultured keratinocytes on the neodermis of INTEGRA(trademark) artificial skin. Second, in addition to acting as a feeder layer, the dermal fibroblasts in the inventive CBSG material evidently produce a number of proteins such as native collagen fibers and fibronectin which is believed to facilitate the attachment of a cultured graft. Third, the cultured keratinocytes of the inventive CBSG can be grafted five to seven days sooner than can traditionally-cultured keratinocytes. This is because cultured keratinocytes of the inventive CBSG are capable of being transferred and grafted at the sub-confluent or less differentiated stage rather than at a later confluent stage. Fourth, since there is minimal need for a donor site there is less likelihood of widespread scarring related to donor site harvesting. Fifth, the cultured keratinocytes of CBSG can be handled much more easily than the conventional CEA during its application on the neodermis of the artificial skin. Fewer cultured cells are lost or damaged during the transfer and application of CBSG. This should improve the success rate of the cultured graft. Sixth, the inventive engraftment technique can result in higher demand and broader scope of clinical applications for artificial skin.
The invention will be better understood by reference to the following detailed description in connection with the accompanying drawings.